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A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis.
The CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In Arabidopsis, constitutive promoters usually result in a low efficiency of heritable mutation in the T1 generation. In this work, CRISPR/Cas9 gene editing efficiencies using different promoters to drive Cas9 expression were evaluated. Expression of Cas9 under the constitutive CaMV 35S promoter resulted in a 2.3% mutation rate in T1 plants and failed to produce homozygous mutations in the T1 and T2 generations. In contrast, expression of Cas9 under two cell division-specific promoters, YAO and CDC45, produced mutation rates of 80.9% to 100% in the T1 generation with nonchimeric mutations in the T1 (4.4⁻10%) and T2 (32.5⁻46.1%) generations. The pCDC45 promoter was used to modify a previously reported multiplex CRISPR/Cas9 system, replacing the original constitutive ubiquitin promoter. The multi-pCDC45-Cas9 system produced higher mutation efficiencies than the multi-pUBQ-Cas9 system in the T1 generation (60.17% vs. 43.71%) as well as higher efficiency of heritable mutations (11.30% vs. 4.31%). Sextuple T2 homozygous mutants were identified from a construct targeting seven individual loci. Our results demonstrate the advantage of using cell division promoters for CRISPR/Cas9 gene editing applications in Arabidopsis, especially in multiplex applications.
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Towards functional genetics in the European amphioxus: Efficient CRISPR/Cas9 editing reveals Ascl1/2.1 requirement for peripheral nervous system development.
Amphioxus, or cephalochordates, have a key phylogenetic position among chordates and serve as pivotal invertebrate models for investigating the evolutionary origins of vertebrate traits. Although functional genetic tools have recently been developed, their application has been limited to the Floridian and Asian species, Branchiostoma floridae and Branchiostoma belcheri, respectively. In this study, we established a CRISPR/Cas9-based genome editing protocol to generate F0 mosaic mutants (crispants) in the European amphioxus B. lanceolatum. As a proof of concept, we targeted the Bl-Ascl1/2.1 gene, a putative regulator of epidermal sensory neuron (ESN) development in the peripheral nervous system coding for a bHLH transcription factor. Using a novel microinjection method of the sgRNA/Cas9 complex in fertilized eggs and two-cell stage embryos, we demonstrated the disruption of Bl-Ascl1/2.1 that resulted in a partial to complete loss of ESNs. Importantly, this phenotype could be rescued by Bl-Ascl1/2.1 mRNA microinjection. These findings demonstrate the efficiency of CRISPR/Cas9-mediated gene editing in B. lanceolatum and establish a foundation for future functional studies in this emerging EvoDevo model.
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[Effect of Knocking Out HOXA5 Gene by CRISPR-Cas9-Mediated Gene Editing Technique on Proliferation of Acute Myeloid Leukemia Cells].
OBJECTIVE: To construct a acute myeloid leukemia (AML) cell line in which HOXA5 gene is stably knocked out by CRISPR-Cas9-mediated gene editing technique, so as to clarify the effect of HOXA5 gene knockout on the proliferation of AML cells, and preliminarily explore the role of HOXA5 gene in the pathogenesis of AML. METHODS: The expression of HOXA5 in bone marrow mononuclear cells (BMMC) of non-tumor hematological patients and newly diagnosed AML patients was detected by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. The AML cell line KO-HOXA5-THP-1 was constructed in which HOXA5 gene was knocked out by CRISPR-Cas9-Mediated gene editing technique, and the knockout of HOXA5 gene was verified by qRT-PCR and Western blot, and the cell proliferation was detected by CCK-8 assay. RESULTS: Compared with non-tumor hematological patients, the levels of HOXA5 gene and protein in BMMC of newly diagnosed AML patients were significantly increased (P <0.05). The stable HOXA5 knockout cell line can be obtained by CRISPR-Cas9-Mediated gene editing technique, and the proliferation ability of THP-1 cells with HOXA5 gene knockout was significantly decreased (P <0.05). CONCLUSION: HOXA5 is highly expressed in AML cells, and knocking out HOXA5 can significantly affect the proliferation ability of AML cells, which provides a new potential therapeutic target for the precise treatment of AML.
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Physiological and Molecular Mechanisms of Lepidopteran Insects: Genomic Insights and Applications of Genome Editing for Future Research.
Lepidopteran insects are a major threat to global agriculture, causing significant crop losses and economic damage. Traditional pest control methods are becoming less effective due to the rapid evolution of insecticide resistance. This study explores the current status and genomic characteristics of 1315 Lepidopteran records, alongside an overview of relevant research, utilizing advanced functional genomics techniques, including RNA-seq and CRISPR/Cas9 gene-editing technologies to uncover the molecular mechanisms underlying insecticide resistance. Our genomic analysis revealed significant variability in genome size, assembly quality, and chromosome number, which may influence species' biology and resistance mechanisms. We identified key resistance-associated genes and pathways, including detoxification and metabolic pathways, which help these insects evade chemical control. By employing CRISPR/Cas9 gene-editing techniques, we directly manipulated resistance-associated genes to confirm their roles in resistance, demonstrating their potential for targeted interventions in pest management. These findings emphasize the value of integrating genomic data into the development of effective and sustainable pest control strategies, reducing reliance on chemical insecticides and promoting environmentally friendly integrated pest management (IPM) approaches. Our study highlights the critical role of functional genomics in IPM and its potential to provide long-term solutions to the growing challenge of Lepidopteran resistance.
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Gene therapy for mitochondrial disorders.
In this review, we detail the current state of application of gene therapy to primary mitochondrial disorders (PMDs). Recombinant adeno-associated virus-based (rAAV) gene replacement approaches for nuclear gene disorders have been undertaken successfully in more than ten preclinical mouse models of PMDs which has been made possible by the development of novel rAAV technologies that achieve more efficient organ targeting. So far, however, the greatest progress has been made for Leber Hereditary Optic Neuropathy, for which phase 3 clinical trials of lenadogene nolparvovec demonstrated efficacy and good tolerability. Other methods of treating mitochondrial DNA (mtDNA) disorders have also had traction, including refinements to nucleases that degrade mtDNA molecules with pathogenic variants, including transcription activator-like effector nucleases, zinc-finger nucleases, and meganucleases (mitoARCUS). rAAV-based approaches have been used successfully to deliver these nucleases in vivo in mice. Exciting developments in CRISPR-Cas9 gene editing technology have achieved in vivo gene editing in mouse models of PMDs due to nuclear gene defects and new CRISPR-free gene editing approaches have shown great potential for therapeutic application in mtDNA disorders. We conclude the review by discussing the challenges of translating gene therapy in patients both from the point of view of achieving adequate organ transduction as well as clinical trial design.
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Exonuclease-fused CRISPR-cas system enhances targeted genome editing for functional genomics in soybean.
CRISPR/Cas technologies have revolutionized plant genome editing, yet their inherent bias toward small insertions or deletions (indels) limits their utility for dissecting regulatory elements and generating impactful allelic variants. Here, we report the development and systematic evaluation of exonuclease-fused CRISPR/Cas systems in soybean to overcome this limitation. We engineered fusions of Cas9 and Cas12a with bacteriophage T5 exonuclease and human TREX2 and assessed their editing performance at the GmWOX5 locus using Agrobacterium rhizogenes-mediated transformation and deep amplicon sequencing. While native Cas9 and Cas12a predominantly generated micro-size deletions (1-10 bp), T5-Exo fusions shifted the mutation spectrum, producing a high frequency of moderate (26-50 bp) and large (> 50 bp) deletions. TREX2 fusions preferentially enhanced the generation of small (11-25 bp) to moderate deletions (26-50 bp). Fusion of exonucleases to Cas9 substantially reduced insertion frequencies and promoted more precise deletion patterns, as observed in T5-Exo-Cas9 and TREX2-Cas9. Deletions from both exonuclease fusions were biased toward the PAM-proximal region, reflecting altered repair outcomes likely driven by directional exonuclease activity and enhanced end resection. These results demonstrate that exonuclease fusions effectively expand the CRISPR toolkit by enabling efficient, targeted generation of larger deletions, which are often required for targeting cis-regulatory elements and microRNAs.
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CRISPR/Cas9-Mediated HY5 Gene Editing Reduces Growth Inhibition in Chinese Cabbage (Brassica rapa) under ER Stress.
Various stresses can affect the quality and yield of crops, including vegetables. In this study, CRISPR/Cas9 technology was employed to examine the role of the ELONGATED HYPOCOTYL 5 (HY5) gene in influencing the growth of Chinese cabbage (Brassica rapa). Single guide RNAs (sgRNAs) were designed to target the HY5 gene, and deep-sequencing analysis confirmed the induction of mutations in the bZIP domain of the gene. To investigate the response of Chinese cabbage to endoplasmic reticulum (ER) stress, plants were treated with tunicamycin (TM). Both wild-type and hy5 mutant plants showed increased growth inhibition with increasing TM concentration. However, the hy5 mutant plants displayed less severe growth inhibition compared to the wild type. Using nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) staining methods, we determined the amount of reactive oxygen species (ROS) produced under ER stress conditions, and found that the hy5 mutant plants generated lower levels of ROS compared to the wild type. Under ER stress conditions, the hy5 mutant plants exhibited lower expression levels of UPR- and cell death-related genes than the wild type. These results indicate that CRISPR/Cas9-mediated editing of the HY5 gene can mitigate growth inhibition in Chinese cabbage under stresses, improving the quality and yield of crops.
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Recent advances in CRISPR-Cas systems for colorectal cancer research and therapeutics.
INTRODUCTION: Colon cancer, ranked as the fourth leading global cause of cancer death, exhibits a complex progression marked by genetic variations. Over the past decade, the utilization of diverse CRISPR systems has propelled accelerated research into colorectal cancer (CRC) treatment. AREAS COVERED: CRISPR/Cas9, a key player in this research, identifies new oncogenes, tumor suppressor genes (TSGs), and drug-resistance genes. Additionally, it facilitates the construction of experimental models, conducts genome-wide library screening, and develops new therapeutic targets, especially for targeted knockout in vivo or molecular targeted drug delivery, contributing to personalized treatments and significantly enhancing the care of colon cancer patients. In this review, we provide insights into the mechanism of the CRISPR/Cas9 system, offering a comprehensive exploration of its applications in CRC, spanning screening, modeling, gene functions, diagnosis, and gene therapy. While acknowledging its transformative potential, the article highlights the challenges and limitations of CRISPR systems. EXPERT OPINION: The application of CRISPR/Cas9 in CRC research provides a promising avenue for personalized treatments. Its potential for identifying key genes and enabling experimental models and genome-wide screening enhances patient care. This review underscores the significance of CRISPR-Cas9 gene editing technology across basic research, diagnosis, and the treatment landscape of colon cancer.
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CRISPR-Cas9-induced genetic mosaicism in three species of the microcrustacean Daphnia.
Genetic mosaicism can arise from in vivo CRISPR-Cas9 gene editing, especially in the embryos. This study evaluates the extent of genetic mosaicism resulted from CRISPR-Cas9-mediated knockout for 11 genes in the freshwater microcrustacean Daphnia magna, Daphnia pulex, and Daphnia sinensis. Based on extensive genotyping data of the asexually produced progenies of successfully edited females, we find strong evidence of mosaicism in 9 of these genes. The genotyping data also suggest that the gene editing activity can take place as early as the one-cell embryo stage and extends into the 32-cell and later stages. This study establishes genetic mosaicism as an important feature of Cas9-mediated gene editing in Daphnia.
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PASTE: a high-throughput method for large DNA insertions.
Prime editing (PE) enables precise genome editing at targeted locus without inducing double-stranded breaks (DSBs). Despite its precision, PE lacks the tendency to integrate large DNA fragments into the genome. Recently, Yarnall et al. reported clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 and an integrase-based system that conducts targeted integration of large DNA sequences (~36 kb) into the genome more efficiently.
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